The main objective of the proposal is to investigate the contribution of cholesterol to the structure and function of plasma membranes and to the growth, proliferation, and malignant transformation of animal cells. The synthesis of cholesterol in normal and neoplastic transformed cells will be inhibited by treatment with oxygenated sterols (e.g., 25-hydroxycholesterol) which specifically depress the regulatory enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The effects of depletion of cellular cholesterol on cellular proliferation and on membrane associated functions such as cell adhesion and cell-to-cell attachment will be examined. The rate of cholesterol synthesis and the activity of HMG-CoA reductase will be assayed during various stages of the cell cycle. Cells will be synchronized by a technique which involves isolating early interphase cells by sucrose density-gradient centrifugation and then cultivating them under an optimal steady state condition. The sensitivity of the synchronous cells to inhibition by oxygenated sterols will be examined throughout the cycle. We will explore the possibility of synchronizing cells by blocking cholesterol synthesis (consequently, cell division) then releasing the restriction by adding cholesterol and its precursor mevalonate. The mechanisms through which cells become resistant to 25-hydroxycholesterol following treatment with mutagens will be studied. The biochemical characteristics of mutant and wild type cells to be investigated include kinetics and thermal stability of HMG-CoA reductase, rate of cholesterol synthesis, transport of 25-hydroxycholesterol, binding of the diol to cellular receptors, and cholesterol concentrations in the plasma membranes. The genetics of the mutations will be studied by the techniques of karyotyping, chromosomes banding, somatic cell hybridization, and metaphase chromosome transfer. The study may facilitate the understanding of the detailed mechanism through which the synthesis of cholesterol is controlled in animal cells.